Insulin-like Growth Factor 2 (IGF2) is a small mitogenic peptide hormone that functions principally during embryonic growth, where its activity is tightly regulated, but is also frequently deregulated in tumours, which predominantly express IGF2 rather than IGF1. Like IGF1, IGF2 exerts its mitogenic affect predominantly by signalling through the IGF1 receptor (IGF1R), but also unlike IGF1 through isoform A of the Insulin receptor, both leading to tyrosine kinase activation and stimulation of both the mitogen-activated protein (MAP) kinase and PKB/AKT signalling.
Many therapies targeting IGF1R have failed in the clinic. This failure may be due to redundancy, as IGF2 still functions through the Insulin receptor. Furthermore, inhibition of IGF1 signalling causes a feedback loop via the pituitary gland, which produces more growth hormone, which in turn tells the liver to produce more IGF1 [1]. Inhibition of IGF2 signalling does not cause such a feedback loop and preferential targeting of IGF2 over IGF1 may reduce negative feedback effects.
The genes Igf2 and Igf2r are imprinted in mammals, and code for the ligand and cell growth promoter, insulin-like growth factor 2 (IGF2) and the mannose 6-phosphate/IGF2 receptor (M6P/IGF2R or IGF2R), respectively [2, 3]. As imprinted genes, both genes are mono-allelically expressed.
Unlike products of other mammalian imprinted genes, IGF2 and M6P/IGF2R are unusual because they specifically bind with high affinity[4-10]. IGF2 binding is specific for domain 11 of the 15 extra-cellular domains of IGF2R. Membrane bound IGF2R acts to negatively regulate free IGF2 levels by receptor internalisation and intra-cellular degradation of IGF2 [2].
Loss of function of Igf2r through disruption of the maternal allele results in Igf2 dependent overgrowth and fatality [11-15]. Loss of function of IGF2R and gain of function of IGF2 through somatic mutation and increased expression, respectively, are also frequently observed in human cancer[16-20]. Conversely, titration of IGF2 via gain of function of Igf2r, for example by bi-allelic expression, leads to an Igf2 dependent growth reduction [21]. The effects of Igf2r on Igf2 dependent growth are thus modified by two fold changes in allelic dosage, suggesting also that the normal capacity of IGF2R to reduce IGF2 bioavailability depends on the affinity of domain 11 for IGF2.
Wild-type IGF2R domain 11 is selective for IGF2 over IGF1 because of a key specific interaction with threonine 19 of IGF2. The IGF2 binding site within domain 11 of human IGF2R consists of a hydrophobic pocket centred on the CD loop, surrounded by polar and charged residues in the AB, FG and HI loops that complement surface charge on IGF2[3]. The exception to this is an otherwise unfavourable charge-charge interaction between E1544 on the AB loop and D23 on IGF2 [6, 22, 23]. Domain 13 interacts with the AB loop of domain 11, breaking this interaction in the full length receptor and thus contributes to the stability of the complex by decreasing the ‘off-rate’ (koff) of the IGF2 interaction compared to domain 11 alone [22]. Mutations in this structurally sensitive AB loop have, however, resulted in isolated domain 11 analogues with increased affinity for IGF2 (e.g. domain 11E1544K, K1545S, L1547V or clone AB3, KD=15 nM) [3, 23], 57. The higher affinity domain 11AB3 AB loop mutant also led to the solution structure of a stable 24.2 kDa complex (IGF2: domain 11AB3)[3]. Domain 11AB3 retains a relatively fixed conformation of the CD loop upon complex formation, and the mutated AB loop moves to accommodate IGF2 helix 1 and the packing of IGF2 residues T16 and F19. The FG loop also repositions between helices 2 and 3 of IGF2 to accommodate burial of IGF2 residue L53 in the domain 11AB3 binding site. All three of these IGF2 residues are critical for IGF2R binding [22, 24]. Both conformational changes also allow the formation of complementary hydrophobic surfaces and support a range of H-bonding and salt bridging interactions with amino acids in the other loops[3].
Importantly, IGF2 binding to domain 11 co-evolved with the evolution of mammals, as in primitive mammals (monotremes) IGF2 binds with ten-fold lower affinity (KD=250-400 μM vs domain 11WT KD=40-60 nM) [3-5, 10]. The structural evolution of domain 11 suggests that the IGF2 binding site has fully evolved in mammals[3].